Protocol to quantitatively assess the structural integrity of Perineuronal Nets ex vivo

Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system diseases including schizophrenia, Alzheimer disease and epilepsy present with qualitative alteration in PNNs, however prior studies failed to quantitatively assess such changes at single PNN level and correlate them with functional changes in disease. We describe a method to quantify the structural integrity of PNNs using high magnification image analysis of Wisteria Floribunda Agglutinin (WFA)-labeled PNNs in combination with cell-type-specific marker such as PV and NeuN. A polyline intensity profile of WFA along the entire perimeter of cell shows alternate segments with and without WFA labeling, indicating the intact chondroitin sulfate proteoglycan (CSPG) and holes of PNN respectively. This line intensity profile defines CSPG peaks, where intact PNN is present, and CSPG valleys (holes) where the PNN is missing. The average number of peaks reflect the integrity of the lattice assembly of PNN. The average size of PNN holes can be readily computed using image analysis software. Furthermore, degradation of PNNs using a bacterial-derived enzyme, Chondroitinase ABC (ChABC), allows to experimentally manipulate PNNs in situ brain slices during which biophysical properties can be assessed by patch-clamp recordings. We describe optimized experimental parameters to degrade PNNs in brain slices before as well as during recordings to study the possible change in function in real time. Our protocols provide effective and appropriate methods to modulate and quantify the PNN's experimental manipulations.